Research Article Gene Expression Changes in Long-Term in Vitro Human Blood-Brain Barrier Models and Their Dependence on a Transwell Scaffold Material
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Hindawi Journal of Healthcare Engineering Volume 2017, Article ID 5740975, 10 pages https://doi.org/10.1155/2017/5740975 Research Article Gene Expression Changes in Long-Term In Vitro Human Blood-Brain Barrier Models and Their Dependence on a Transwell Scaffold Material 1 1 2 3 Joel D. Gaston, Lauren L. Bischel, Lisa A. Fitzgerald, Kathleen D. Cusick, 4 2 Bradley R. Ringeisen, and Russell K. Pirlo 1American Society for Engineering Education Postdoctoral Fellowship Program, U.S. Naval Research Laboratory, Washington, DC, USA 2Chemistry Division, U.S. Naval Research Laboratory, Washington, DC, USA 3National Research Council Postdoctoral Fellowship Program, U.S. Naval Research Laboratory, Washington, DC, USA 4Defense Advanced Research Projects Agency, Arlington, VA, USA Correspondence should be addressed to Russell K. Pirlo; [email protected] Received 20 June 2017; Revised 19 September 2017; Accepted 12 October 2017; Published 29 November 2017 Academic Editor: George Stan Copyright © 2017 Joel D. Gaston et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Disruption of the blood-brain barrier (BBB) is the hallmark of many neurovascular disorders, making it a critically important focus for therapeutic options. However, testing the effects of either drugs or pathological agents is difficult due to the potentially damaging consequences of altering the normal brain microenvironment. Recently, in vitro coculture tissue models have been developed as an alternative to animal testing. Despite low cost, these platforms use synthetic scaffolds which prevent normal barrier architecture, cellular crosstalk, and tissue remodeling. We created a biodegradable electrospun gelatin mat “biopaper” (BP) as a scaffold material for an endothelial/astrocyte coculture model allowing cell-cell contact and crosstalk. To compare the BP and traditional models, we investigated the expression of 27 genes involved in BBB permeability, cellular function, and endothelial junctions at different time points. Gene expression levels demonstrated higher expression of transcripts involved in endothelial junction formation, including TJP2 and CDH5, in the BP model. The traditional model had higher expression of genes associated with extracellular matrix-associated proteins, including SPARC and COL4A1. Overall, the results demonstrate that the BP coculture model is more representative of a healthy BBB state, though both models have advantages that may be useful in disease modeling. 1. Introduction considerable physical space, and often do no not translate well to human results [2, 3]. One way to overcome the short- The unique characteristics of the blood-brain barrier (BBB) comings of in vivo systems is through in vitro models capable present a considerable challenge for studying central nervous of accurately mimicking human systems. In vitro model sys- system (CNS) therapeutics and disease modeling. Lipid- tems consisting of cell-based arrays are a valuable tool to soluble molecules and gases readily diffuse across the BBB screen molecular BBB permeability prior to animal testing. while larger, hydrophilic molecules are impeded by endothe- The most common platform for in vitro BBB testing lial tight junctions [1]. The intrinsic difficulty of crossing the incorporates multiple cell types cultured on transwell inserts, BBB necessitates extensive testing for new drug candidates allowing for separate culture compartments within the same using relevant in vitro and in vivo models. In vivo model well [1]. The transwell inserts consist of a semiporous poly- organisms, such as rats, provide valuable information in drug carbonate or polyethylene terephthalate (PET) membrane. screening assays, particularly with respect to complex effects Endothelial cells are grown on one side of the membrane, in the natural CNS environment. However, they are expen- and astrocytes are grown on the other, creating a simple bar- sive and time consuming, face ethical concerns, require a rier similar to the natural BBB environment. This basic 2 Journal of Healthcare Engineering model setup provides an approximation of the native BBB with barrier integrity and overall cell function. Here, we microenvironment but has several significant shortcomings describe the analysis of a 27-gene panel, consisting of genes compared to in vivo models. Membrane material, cell-cell associated with barrier integrity and endothelial cell or astro- contact, and fluid shear stress are some of the factors poorly cyte function. Prior to the gene expression panel, the suitabil- addressed by the standard transwell model. Two of these ity of multiple genes to serve as reference genes was examined issues, membrane material and cell-cell contact, can be to ensure an accurate comparison between the models. addressed simultaneously by changing the transwell insert membrane material. 2. Experimental Procedure Transwell insert membranes are a necessary artifact of barrier culture models, where a substrate is essential for cell 2.1. Cell Culture. Primary human astrocytes (HA) and cul- seeding, attachment, and monolayer formation. Synthetic ture media were obtained from ScienCell Research Laborato- barriers, such as polycarbonate or PET, are not readily ries (Carlsbad, CA, USA) and maintained according to the degraded or remodeled by either endothelial cells or astro- manufacturer’s instructions. Primary human brain micro- cytes, making it an unyielding barrier to “normal” BBB archi- vascular endothelial cells (HBMEC) and culture media were tecture. Without degradation, the membrane also imposes a obtained from Cell Systems (Kirkland, WA, USA) and main- hard limit on the degree of endothelial-to-astrocyte cell- tained according to the manufacturer’s instructions. Media cell contact. It has been repeatedly demonstrated that for both cell types contained 10% FBS. Cells used in all exper- proper gene expression and regulation of BBB proteins iments were at passage 1 when seeded. require direct contact between endothelial cells and astro- cytes [4, 5]. Additionally, a monolithic polymer barrier 2.2. Biopaper Insert Fabrication. Cell culture inserts with a between the cell types prevents proper extracellular matrix 15% electrospun gelatin biopaper membrane were fabricated (ECM) deposition and basement membrane formation, a fac- as previously described [8]. Briefly, 10 mL of a gelatin- tor known to affect BBB permeability [6, 7]. Taken together, genipin solution was prepared by dissolving 1.8 grams of type these conclusions point to the need for a biodegradable mem- B gelatin from bovine skin (Sigma-Aldrich, Saint Louis, MO, brane for in vitro models capable of supporting cell growth USA) into 70% (vol%) acetic acid (Sigma-Aldrich) in distilled and remodeling. water. The gelatin solution was magnetically stirred for 60 Previous research replaced the transwell PET membrane minutes before a solution consisting of 0.05 grams of genipin with an electrospun gelatin scaffold, styled “biopaper,” in an (Wako Chemicals, Richmond, VA, USA) dissolved in 0.5 mL endothelial/astrocyte coculture in vitro model [8]. This 21- of ethanol and 1 mL of 1X phosphate-buffered saline (PBS) day study demonstrated that the biopaper coculture models was added to the solution. The homogenous solution was were initially more permeable than standard PET models, then transferred to a 20 mL plastic syringe (Thermo Fisher at least until day 14. At later time points, the biopaper model Scientific, Rockwood, TN, USA) with a 22-gauge stainless was less permeable than the PET, as proven by both transen- steel blunt-ended needle (Jensen Global, Santa Barbara, CA, dothelial electrical resistance (TEER) measurements and USA). The spinning solution was stored between 24 and 72 dextran diffusion across the barrier. Notably, cell morphol- hours until electrospinning. ogy was similar in both models, indicating that a deeper look Gelatin nanofiber biopapers were electrospun using a into the gene expression profiles may be necessary to deter- Matsusada Precision AC100-240V high-voltage power sup- mine differences in transcript expression of essential BBB ply (Kusatsu city, Shiga, Japan) and a New Era Pump NE- proteins between the two models. 300 syringe pump (Farmingdale, NY, USA). The power To date, cellular gene expression profiling of in vitro BBB supply electrode was connected to the stainless steel models has been limited to only a handful of genes relevant to blunt-ended needle and positioned 15 cm from a grounded barrier permeability. Recent coculture models typically only circular stainless steel plate (10 cm diameter) covered with assay 5–10 genes, primarily focusing on endothelial cell junc- nonstick aluminum foil. The syringe pump was set to a tion proteins, with studies lasting for several days [9–11]. flow rate of 5 μL/min, and a voltage of 15 kV was applied Furthermore, several recent studies have employed the use to the electrode for a total of 30 minutes to produce each of immortalized cell lines or nonhuman animal cell lines. electrospun mat, designated “biopapers.” Electrospun bio- While this data is important for improving BBB models, gene papers were placed in a Sanplatec Dry Keeper desiccator expression profiles obtained from primary human cell cocul- (Kita-ku, Osaka, Japan) for at least 24 hours. ture models would be invaluable. Furthermore, an extensive Biopapers were cut to size and placed over the opening